A widely used method for producing hybrid seeds involves crossing a cytoplasmic male sterile (CMS) plant line with a fertile plant line. Typically, the fertile line contains a fertility restorer gene in its nuclear genome, so that all of the progeny are male fertile. All seeds collected from a CMS plant must result from cross-pollination. However, the hybrid seed so generated will itself be male sterile unless the male parent has brought a nuclear fertility-restorer gene into the next generation. The fertility of the progeny is important for productivity in plant varieties where self-pollination is responsible for production of the desirable crop. For example, a fruit crop of a self-pollinated species requires male fertility, while an ornamental species will produce attractive flowers or plant morphology even when no pollen is produced.
While a number of naturally occurring CMS/restorer systems exist and are currently in use for hybrid seed production, there are a number of crop species which lack known CMS and fertility restorer genes. For example, a hybrid seed of tomato is typically made by hand emasculation of plants to be used as female parents. This hand-made method of cross-pollination is quite labor intensive and cost-prohibitive for many crops. In addition, certain naturally occurring CMS/restorer systems have some drawbacks. For example, corn plants carrying the CMS-T cytoplasm are more susceptible to a blight disease.
Fertility restorer genes that have been particularly useful for hybrid seed production are active as single dominant alleles at a locus, though multigenic systems are sometimes used. A Petunia fertility restorer locus termed Rf is known to be effective with no additional helper genes to restore fertility (Edwardson et al., “Fertility Restoration in Cytoplasmic Male Sterile Petunia,” J. Hered., 58:195–196 (1967); Izhar, “Cytoplasmic Male Sterility in Petunia. III. Genetic Control on Microsporogenesis and Male Fertility Restoration,” J. Hered., 69:22–26 (1978)).
Nuclear fertility restoration genes confer normal pollen development upon plants carrying sterility-encoding mitochondria. The mitochondrial genes responsible for causing the male sterility have been identified in a number of species, including Petunia, maize, Brassica, and common bean. The expression of these CMS-encoding mitochondrial genes is affected by the nuclear restorer genes, as shown for Rf in Petunia (Pruitt et al., “Cytochrome Oxidase Subunit II Sequences in Petunia Mitochondria: Two Intron-Containing Genes and an Intron-Less Pseudogene Associated With Cytoplasmic Male Sterility,” Curr. Genet., 16:281–91 (1989); Nivison et al., “Identification of a Mitochondrial Protein Associated With Cytoplasmic Male Sterility in Petunia,” Plant Cell, 1:1121–30 (1989); Nivision et al., “Sequencing, Processing, and Localization of the Petunia CMS-Associated Mitochondrial Protein,” Plant J., 5:613–623 (1994); Hanson et al., “Mitochondrial Gene Organization and Expression in Petunia Male Fertile and Sterile Plants,” J. Hered., 90:362–368 (1999)); Rf1 in CMS-T maize (Dewey et al., “Novel Recombinations in the Maize Mitochondrial Genome Produce a Unique Transcriptional Unit in the Texas Male-Sterile Cytoplasm,” Cell, 44:439–49 (1986); Wise et al., “Mitochondrial Transcript Processing and Restoration of Male Fertility in T-Cytoplasm Maize,” J Hered, 90:380–385 (1999); Kennell et al., “Influence of Nuclear Background on Transcription of a Maize Mitochondrial Region Associated With Texas Male Sterile Cytoplasm,” Mol. Gen. Genet., 210:399–406 (1987); Kennell et al., “Initiation and Processing of atp6, T-urf13, and ORF221 Transcripts From Mitochondria of T Cytoplasm Maize,” Mol. Gen. Genet., 216:16–24 (1989)); Rfp1 and rfp1 in Brassica (Singh et al., Suppression of Cytoplasmic Male Sterility by Nuclear Genes Alters Expression of a Novel Mitochondrial Gene Region,” Plant Cell, 3:1349–1362 (1991); Singh et al., “Nuclear Genes Associated With a Single Brassica CMS Restorer Locus Influence Transcripts of Three Different Mitochondrial Gene Regions,” Genetics, 143:505–516 (1996)); restorers in radish (Krishnasamy et al., “Organ-Specific Reduction in the Abundance of a Mitochondrial Protein Accompanies Fertility Restoration in Cytoplasmic Male-Sterile Radish,” Plant Molec. Biol., 26:935–946 (1994)); restorers in sunflower (Horn et al., “A Mitochondrial 16 kDa Protein is Associated With Cytoplasmic Male Sterility in Sunflower,” Plant Molec. Biol., 17:29–36 (1991); Laver et al., “Mitochondrial Genome Organization and Expression Associated With Cytoplasmic Male Sterility in Sunflower (Helianthus annuus),” Plant J., 1:185–193 (1991); Monéger et al., “Nuclear Restoration of Cytoplasmic Male Sterility in Sunflower is Associated With the Tissue-Specific Regulation of a Novel Mitochondrial Gene,” EMBO J., 13:8–17 (1994); Smart et al., “Cell-Specific Regulation of Gene Expression in Mitochondria During Anther Development in Sunflower,” Plant Cell, 6:811–825 (1994)); restorers in rice (Akagi et al., “A Unique Sequence Located Downstream From the Rice Mitochondrial atp6 May Cause Male Sterility,” Curr. Genet., 25:52–58 (1994); Kadowaki et al., “A Chimeric Gene Containing the 5′ Portion of atp6 is Associated With Cytoplasmic Male Sterility of Rice,” Mol. Gen. Genet., 224:10–16 (1990)); and Fr2 in broad bean (Chase, “Expression of CMS-Unique and Flanking Mitochondrial DNA Sequencs in Phaseolus vulgaris,” L. Curr. Genet., 25:245–251 (1993); He et al., “Pollen Fertility Restoration by Nuclear Gene Fr in CMS Bean: Nuclear-Directed Alteration of a Mitochondrial Population,” Genetics, 139:995–962 (1995)). The expression of various nuclear restorer genes has been reported to be either enhanced in reproductive tissue, as in the case of sunflower, or, as in the case of Petunia, expressed in both vegetative and reproductive tissues. Thus, different fertility restorer genes carry different promoters and nuclear expression regulatory elements which may confer very limited tissue-specific expression or very broad expression in the plant.
Reduction in the amount of the protein product of the CMS-encoding gene is the usual effect of these restorers whose target mitochondrial genes are known. These genes may possibly act by affecting the transcription or translation rate, the transcript or protein stability, processing, splicing, etc. Alleles of some restorer genes may up-regulate while others may down-regulate the expression of particular mitochondrial genes. Fertility restorer genes and their alleles or homologous counterparts in other species may thus be extremely valuable in engineering the expression of genes introduced into higher plant mitochondria.
The cloning and sequencing of the restorer gene Rf2 in maize has been reported in Cui et al., “The rf2 Nuclear Restorer Gene of Male-Sterile T-Cytoplasm Maize,” Science, 272:1334–1336 (1996) and U.S. Pat. No. 5,981,833 to Wise et al. This restorer gene acts in conjunction with a second required gene, Rf1, the gene that reduces the amount of the toxic protein, to restore fertility to plants carrying the maize CMS-T cytoplasm (Dewey et al., “Novel Recombinations in the Maize Mitochondrial Genome Produce a Unique Transcriptional Unit in the Texas Male-Sterile Cytoplasm,” Cell, 44:439–49 (1986); Dewey et al., “A Mitochondrial Pprotein Associated With Cytoplasmic Male Sterility in the T Cytoplasm of Maize,” Proc. Natl. Acad. Sci. USA, 84:5374–78 (1987); Wise et al., “Urf13-T of T Cytoplasm Maize Mitochondria Encodes a 13 kD Polypeptide,” Plant Mol. Biol. 9:121–26 (1987)). Plants of genotype Rf1rf2, though sterile, have greatly reduced amounts of the URF13 protein. In contrast, sterile plants of genotype rf1Rf2 have abundant amounts of the URF13 protein. The Rf2 gene is, thus, unusual in that no effect on the expression of the maize T-CMS-associated protein, URF13, has been detected. The sequence of the gene bore out the absence of observable effect on mitochondrial gene expression; according to sequence analysis, Rf2 is apparently an aldehyde dehydrogenase (Liu et al., “Mitochondrial Aldehyde Dehydrogenase Activity is Required for Male Fertility in Maize,” The Plant Cell, 13:1063–1078 (2001)). It has been proposed that Rf2 acts by compensating for a metabolic defect caused by the low levels of the URF13 protein that remain despite the presence of Rf1, the gene that reduces the amount of the toxic protein (Dewey et al., “A Mitochondrial Protein Associated With Cytoplasmic Male Sterility in the T Cytoplasm of Maize,” Proc. Natl. Acad. Sci. USA, 84:5374–78 (1987)) and also alters the T-urf13 transcript profile (Kennell et al., “Influence of Nuclear Background on Transcription of a Maize Mitochondrial Region Associated With Texas Male Sterile Cytoplasm,” Mol. Gen. Genet., 210:399–406 (1987)).
An abnormal recombinant mitochondrial gene in Petunia CMS lines (termed pcf) has been genetically correlated with CMS (Young et al., “A Fused Mitochondrial Gene Associated With Cytoplasmic Male Sterility is Developmentally Regulated,” Cell, 50:41–49 (1987)). Because plant mitochondrial RNA is edited from C to U in some locations, the edited RNA sequence for the pcf gene has been determined, allowing the prediction of the pcf-encoded protein (Wintz et al., “A Termination Codon is Created by RNA Editing in the Petunia Mitochondrial atp9 Gene Transcript,” Curr. Genet., 19:61–64 (1990); Sutton et al., “Editing of Pre-mRNAs Can Occur Before cis- and trans-Splicing in Petunia Mitochondria,” Mol. Cell Biol., 11:4274–4277 (1991); Nivision et al., “Sequencing, Processing, and Localization of the Petunia CMS-Associated Mitochondrial Protein,” Plant J., 5:613–623 (1994); Hanson et al., “Mitochondrial Gene Organization and Expression in Petunia Male Fertile and Sterile Plants,” J. Hered., 90:362–368 (1999)). Antibodies to synthetic peptide sequences have revealed the presence of a 19.5 kD PCF protein located in both the membrane and soluble fraction of mitochondria (Nivison et al., “Identification of a Mitochondrial Protein Associated With Cytoplasmic Male Sterility in Petunia,” Plant Cell, 1:1121–30 (1989)). The PCF protein is processed from a longer precursor protein and is entirely encoded by the urfS region of the pcf gene (Nivision et al., “Sequencing, Processing, and Localization of the Petunia CMS-Associated Mitochondrial Protein,” Plant J., 5:613–623 (1994)). The PCF protein is strongly expressed in sporogenous cells of premeiotic petunia anthers in CMS lines, but undetectable in CMS-Rf lines (Conley et al., “Tissue-Specific Protein Expression in Plant Mitochondria,” Plant Cell, 6:85–91 (1994)). Abnormalities in Petunia pollen development are first observed in meiosis, and by the developmental stage where fertile plants are releasing pollen, CMS anthers are hollow shells (Conley et al., “Effects of Petunia Cytoplasmic Male Sterile (CMS) Cytoplasm on the Development of Sterile and Fertility-Restored P. parodii Anthers,” Am. J. Bot., 81:630–640 (1994)). It is evident that the pcf gene product is disrupting mitochondrial function, leading to death of the sporogenous cells, though the exact mechanism at the molecular level is not known.
In maize T, Petunia, rice, and Brassica Pol CMS systems, particular transcripts of CMS-associated genes have been reported to be altered in restored lines (Pruitt et al., “Transcription of the Petunia Mitochondrial CMS-Associated pcf Locus in Male Sterile and Fertility-Restored Lines,” Mol. Gen. Genet., 227:348–355 (1991); Dewey et al., “Novel Recombinations in the Maize Mitochondrial Genome Produce a Unique Transcriptional Unit in the Texas Male-Sterile Cytoplasm,” Cell, 44:439–49 (1986); Kennell et al., “Initiation and Processing of atp6, T-urf13, and ORF221 Transcripts From Mitochondria of T Cytoplasm Maize,” Mol. Gen. Genet., 216:16–24 (1989); Kennell et al., “Influence of Nuclear Background on Transcription of a Maize Mitochondrial Region Associated With Texas Male Sterile Cytoplasm,” Mol. Gen. Genet., 210:399–406 (1987); Singh et al., “Suppression of Cytoplasmic Male Sterility by Nuclear Genes Alters Expression of a Novel Mitochondrial Gene Region,” Plant Cell, 3:1349–1362 (1991); Singh et al., “Nuclear Genes Associated With a Single Brassica CMS Restorer Locus Influence Transcripts of Three Different Mitochondrial Gene Regions,” Genetics, 143:505–516 (1996); Wise et al., “Mitochondrial Transcript Processing and Restoration of Male Fertility in T-Cytoplasm Maize,” J. Hered., 90:380–385 (1999)). In Brassica, the presence of either one of two restorer genes results in monocistronic transcripts of atp6, instead of the dicistronic orf224/atp6 transcripts found in CMS lines (Singh et al., “Suppression of Cytoplasmic Male Sterility by Nuclear Genes Alters Expression of a Novel Mitochondrial Gene Region,” Plant Cell, 3:1349–1362 (1991)). A UG-rich sequence appears to be the target of the Brassica restorer alleles (Singh et al., “Nuclear Genes Associated With a Single Brassica CMS Restorer Locus Influence Transcripts of Three Different Mitochondrial Gene Regions,” Genetics, 143:505–516 (1996)). In Petunia, pcf transcripts with 5′ termini at −121 are specifically reduced in restored lines (Pruitt et al., “Transcription of the Petunia Mitochondrial CMS-Associated pcf Locus in Male Sterile and Fertility-Restored Lines,” Mol. Gen. Genet., 227:348–355 (1991)), while transcripts terminating at −266 and −522 remain at normal levels. In maize T cytoplasm, a sequence unlike either the Brassica restorer target or the Petunia −121 transcript terminus is the putative recognition signal for the Rf1 gene (Dill et al., “Rf8 and Rf* Mediate Unique T-urf13-Transcript Accumulation, Revealing a Conserved Motif Associated With RNA Processing and Restoration of Pollen Fertility in T-cytoplasm Maize,” Genetics, 147:1367–1379 (1997)).
The steady-state amounts of the Petunia pcf-encoded protein and the maize urf13-encoded protein decrease greatly in restored lines compared to unrestored lines (Nivison et al., “Identification of a Mitochondrial Protein Associated With Cytoplasmic Male Sterility in Petunia,” Plant Cell, 1:1121–30 (1989); Dewey et al., “Novel Recombinations in the Maize Mitochondrial Genome Produce a Unique Transcriptional Unit in the Texas Male-Sterile Cytoplasm,” Cell, 44:439–49 (1986); Wise et al., “Urf13-T of T Cytoplasm Maize Mitochondria Encodes a 13 kD Polypeptide,” Plant Mol. Biol., 9:121–26 (1987)). Abundance of CMS-associated proteins is also reduced in sunflower and radish (Horn et al., “A Mitochondrial 16 kDa Protein is Associated With Cytoplasmic Male Sterility in Sunflower,” Plant Mol. Biol. 17:29–36 (1991); Laver et al., “Mitochondrial Genome Organization and Expression Associated With Cytoplasmic Male Sterility in Sunflower (Helianthus annuus),” Plant J., 1:185–193 (1991); Krishnasamy et al., “Organ-Specific Reduction in the Abundance of a Mitochondrial Protein Accompanies Fertility Restoration in Cytoplasmic Male-Sterile Radish,” Plant Mol. Biol., 26:935–946 (1994)). The mechanism behind the reduction in quantity of CMS-associated proteins in restored lines is not understood. For example, absence of transcripts that could potentially encode the PCF protein is not the explanation; only the shortest transcript is reduced in restored lines (Pruitt et al., “Transcription of the Petunia Mitochondrial CMS-Associated pcf Locus in Male Sterile and Fertility-Restored Lines,” Mol. Gen. Genet., 227:348–355 (1991)).
In Petunia and in some other CMS/restorer systems, the abnormal gene is co-transcribed with known mitochondrial genes. One possible mechanism for CMS in Petunia and its restoration, which is also consistent with current data, is that the restorer gene not only results in decrease in the expression of PCF, but also improves the expression of the co-transcribed genes nad3 and rps12 in some way. For example, it remains possible that an RNA processing event results in little translation of PCF but enhanced production of NAD3 and RPS12 protein.
In sum, with the exception of maize Rf2, in those systems where analysis has reached the molecular level, restorer genes have been found to affect the abundance of mitochondrial-encoded DNAs, RNAs, and proteins.
Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have been cloned.
The present invention is directed to overcoming these deficiencies in the art.